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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Produce Safety and Microbiology Research » Research » Publications at this Location » Publication #345153

Research Project: Molecular Identification and Characterization of Bacterial and Viral Pathogens Associated with Foods

Location: Produce Safety and Microbiology Research

Title: Proteolytic surface-shaving and serotype-dependent expression of SPI-1 invasion proteins in Salmonella enterica subspecies enterica

item Fagerquist, Clifton - Keith
item Zaragoza, William

Submitted to: Frontiers in Nutrition
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/23/2018
Publication Date: 12/10/2018
Citation: Fagerquist, C.K., Zaragoza, W.J. 2018. Proteolytic surface-shaving and serotype-dependent expression of SPI-1 invasion proteins in Salmonella enterica subspecies enterica. Frontiers in Nutrition. 5:124.

Interpretive Summary: The outer envelope of a bacterial cell (whether membrane or cell wall) is the primary barrier (as well as interface) by which the microorganism interacts with its environment as well as any host it may happen to infect. As such, characterizing the outer bacterial surface can provide useful information about the microorganism as well as clues to its virulence in the case of pathogenic bacteria. This information, once obtained, can be used to develop targets for new vaccines as well as detection assays (e.g. monoclonal antibodies) that function by recognizing unique surface features. Surface-shaving is a technique by which surface-exposed biomolecules (usually proteins) are cleaved from the bacterial cell surface by enzymes and subsequently identified by liquid chromatography tandem mass spectrometry (LC-MS/MS). Proteolytic enzymes, such as trypsin, are used to cleave surface-exposed proteins. We have used this technique on three serovars/strains of Salmonella eneterica enterica (SEE): Newport, Kentucky and Thompson. We detected significant evidence for proteolytic flagella damage as well as evidence of up-regulation of flagella biosynthesis. Flagella play a critical role in the mobility, survival and virulence of SEE. Most significantly, secreted effector/invasion proteins were also detected for SEE Newport and Thompson strains. Exposure to trypsin appeared to trigger a rapid (< 15 minutes) biological invasion response. These effector/invasion proteins were not detected in the SEE Kentucky strain. It remains to be determined as to how SEE senses proteolytic damage to its flagella and whether this “sense” also results in certain SEE serovars mounting an invasion response.

Technical Abstract: We performed proteolytic surface-shaving with trypsin on three strains/sevovars of Salmonella enterica enterica (SEE): Newport, Kentucky and Thompson. Surfaced-exposed proteins of live bacterial cells were digested for fifteen minutes. A separate twenty hour re-digestion was also performed on the supernatant of each shaving experiment to more completely digest protein fragments into detectable peptides for proteomic analysis by nano-liquid chromatography-electrospray ionization-Orbitrap mass spectrometry. Control samples (i.e. no trypsin during surface-shaving step) were also performed in parallel. We detected peptides of flagella proteins: FliC (filament), FliD (cap) and FlgL (hook-filament junction) as well as peptides of FlgM (anti-sigma28 factor), i.e. the negative regulator of flagella synthesis. For SEE Newport and Thompson, we detected Salmonella pathogenicity island 1 (SPI-1) secreted effector/invasion proteins: SipA, SipB, SipC and SipD, whereas no Sip proteins were detected in control samples. No Sip proteins were detected for SEE Kentucky (or its control) although sip genes were confirmed to be present. Our results may suggest a biological response (< 15 minutes) to proteolysis of live cells for these SEE strains and, in the case of Newport and Thompson, a possible invasion response.